Integrative taxonomy and multigene phylogeny uncover three novel taxa of the genus Clitopilus from southern Punjab, Pakistan

In this study, we employ integrative taxonomy and multigene phylogenetic analysis to identify and describe three novel species of the genus Clitopilus from southern Punjab, Pakistan. Detailed morphological examinations were conducted alongside advanced phylogenomic techniques, incorporating sequences from multiple genetic loci, including internal transcribed spacer (ITS), large ribosomal subunit (28S), and second largest of RNA polymerase-II (RPB2) regions. Our comprehensive morphological and phylogenetic approach allowed us to delineate distinct morphological traits and unique genetic profiles of the newly identified taxa viz. C. albominimus, C. inops, and C. pirosylvanus. The multigene phylogenetic analysis using maximum likelihood and Bayesian inference methods showed that the three new species form distinct branches, separate from any previously reported Clitopilus species, and placed them within the Clitopilus sect. Scyphoides. This research underscores the importance of integrative methods combining classical taxonomy with genomic tools to elucidate fungal biodiversity. Our findings not only expand the known diversity of Clitopilus in Pakistan but also provide valuable insights into the evolutionary relationships within the genus, paving the way for future studies on their ecological roles and conservation needs.